For many short-lived eukaryotic proteins, covalent attachment to the polypeptide ubiquitin is a prerequisite for their degradation; examples include the destruction of cyclins in cell cycle control (Glotzer et al., 1991) and degradation of proteins that regulate development, e.g., the vertebrate c-mos protein kinase (Okazaki et al., 1992) or the yeast MAT.alpha.2 transcription factor (Hochstrasser et al., 1991; Chen et al., 1993). The ubiquitin system is essential for phenomena as diverse as the heat shock response and DNA repair, and both ubiquitin and the enzymes involved in ubiquitin metabolism are highly conserved among eukaryotic phyla (Finley and Chau, 1991; Hershko and Ciechanover, 1992; Hockstrasser et al., 1992; Jentsch, 1992; Varshavsky, 1992). Using a MAT.alpha.2 derivative as substrate, a ubiquitin-dependent degradation pathway in the yeast Saccharomyces cerevisiae, the DOA (degradation of alpha) pathway has recently been identified (Chen et al., 1993; Hochstrasser & Varshavsky, 1990). This pathway, which requires two ubiquitin-conjugating (Ubc) enzymes, Ubc6 (Doa2) and Ubc7, targets .alpha.2 via an element within the first 67 residues of the .alpha.2 repressor, the Deg1 degradation signal. Many more gene products have been implicated in .alpha.2 degradation, based on genetic analyses (Chen et al., 1993).
Deubiquitinating enzymes serve a number of functions (Hochstrasser, 1992; Rose, 1988). First, ubiquitin must be cleaved from a set of biosynthetic precursors, which occur either as a series of ubiquitin monomers in head-to-tail linkage or as fusions to certain ribosomal proteins (Finley & Chau, 1991). Secondly, ubiquitin must be recycled from intracellular conjugates, both to maintain adequate pools of free ubiquitin and, in principle at least, to reverse the modification of inappropriately targeted proteins. Finally, deubiquitinating reactions may be integral to the degradation of ubiquitinated proteins by the 26S proteasome, a complex ATP-dependent enzyme whose exact composition and range of activities remain poorly characterized (Hershko & Ciechanover, 1992; Hardari et al., 1992; Murakami, 1992; Rechsteiner, 1993).
Consistent with these diverse functions, eukaryotic cells have been shown to contain many distinct deubiquitinating enzymes (Rose, 1988; May & Wilkinson, 1989; Jonnalagadda, 1989; Baker et al., 1992). Previously, four deubiquitinating enzyme genes, YUH1, UBP1, UBP2, and UBP3, had been identified in S. cerevisiae (Baker et al., 1992; Miller, 1989; Tobias & Varshavsky, 1992). A mutant strain lacking all four genes grows normally and shows continued high deubiquitinating activity (Baker et al., 1992).